Development of genetic recombination technologies has provided a variety of protein pharmaceuticals. In particular, numerous antibody and protein pharmaceuticals have been recently developed and commercialized. To prepare these protein pharmaceuticals in an industrial and economic manner and in a high purity has become a more important issue in biopharmaceutical industry.
Generally, these antibody or protein pharmaceuticals are produced by culturing recombinant cells into which a vector including an objective protein gene is inserted. The culture fluid includes impurities such as various medium components, cell by-products or the like, in addition to the objective protein. Thus, it is very difficult and challenging to perform isolation and purification to meet purity requirements for pharmaceuticals, and further combine the industrial-scale production and economic efficiency.
In the preparation method of the objective protein, the purification process is a very important process in increasing purity of the protein while maintaining its physiological functions. In general, the purification process is carried out by a combination of different modes of chromatography based on differences in charge, hydrophilicity or molecular size, and a manipulation such as alcohol fractionation, salting-out, filtration, concentration or dilution, and the like (Non-Patent Document 1).
Chromatography may be exemplified by affinity chromatography, cation exchange chromatography, anion exchange chromatography, hydroxyapatite chromatography, reversed-phase chromatography, hydrophobic interaction chromatography, size exclusion chromatography, mixed mode chromatography and the like. A combination thereof can be carried out to purify the objective protein with a desired purity. In affinity chromatography, a carrier, on which a substance having affinity for the objective protein such as antibody or heparin is immobilized, is used.
If the objective protein is an antibody, a carrier, on which a ligand (affinity substance) interacting with a specific region of the antibody such as Protein A or Protein G is immobilized, is used. Further, considering the interaction between the carrier or buffer used in these chromatographies and objective protein and impurities, two methods can be employed; a capture mode for adsorbing the objective protein onto the carrier and then eluting it, and a flow-through mode for adsorbing impurities onto the carrier and then passing the objective protein through the carrier.
In particular, if the objective protein is an antibody, it has been already established to purify an object with a desired purity by Protein A affinity chromatography and by combinations of one or more of the chromatographies described above, and this technology has been used as a platform purification method.
Meanwhile, if the objective protein is proteins other than antibodies, there is no standard method as the platform purification method of the protein with the desired purity. Considering the characteristics of the objective protein, a purification process is designed by repeating a test by trial and error respectively, and optimized for each objective protein respectively, and then practically applied.
Affinity chromatography using an antibody having an affinity for the objective protein has been also used for purification. However, there are many problems that a ligand (affinity substance) specific to the objective protein must be used, it is no always easy to obtain a ligand having a desired binding property or affinity, a carrier binding with the ligand is considerably expensive, and there is a concern for its stable supply. Therefore, it is not easy to achieve its industrial application.
Under this background, there is a need to establish a standard technology capable of purifying the objective protein in a simple, efficient, and rapid manner.
Meanwhile, when a simple chromatography other than affinity chromatography is used, pretreatment of cell culture supernatant or the like is essential for purification of the objective protein. Typically, the cell culture supernatant includes a large amount of cell metabolites or medium components, and components derived from additives during cultivation, and the cell culture supernatant has high conductivity or salt concentration in many cases.
In this regard, it is not efficient to directly load the culture supernatant to the chromatography, and thus a pretreatment such as several-fold dilution of the culture supernatant, exchange of a buffer, exchange of the buffer after concentration, dilution after concentration or the like are typically performed.
However, because such pretreatments require several hours to 1 day or longer of manipulation time, and depend on production facility such as size of dilution tank or the like, the production methods are not efficient and practical in terms of industrial application. Further, quality of the objective protein may deteriorate during pretreatment of the culture supernatant such as concentration or the like.
As such, the pretreatment manipulation becomes a challenging issue in the industrial production method of the protein with a high purity. There is a need for a purification method capable of efficiently recovering the objective protein by a simple pretreatment manipulation without damaging quality of the protein.
Further, in order to supply and use the objective protein as a medicine, it is required that the objective protein is highly purified from a composition such as culture fluid in order to have a desired biological activity. However, most of the compositions containing the objective protein as a starting point of purification, like culture fluid, include trace elements having a proteolytic activity or impurities. If these components are not inactivated or removed, there are concerns for damage in the biological activity of the objective protein, a reduction in the yield, modification, production of by-products, or the like. In addition, the impurities are concentrated or activated during the above described pretreatment of the culture supernatant, which may deteriorate the objective protein.
The components or impurities may be exemplified by glycolytic enzymes, proteolytic enzymes, oxidoreductases and the like which are retained within the producing cells. Examples of the glycolytic enzymes may include neuraminidase (sialidase), galactosidase, glycanase and the like.
Examples of the proteolytic enzymes may include serine protease, esterase, cysteine protease, trypsin-like protease, aminopeptidase, aspartic protease, cathepsin and the like.
Examples of the oxidoreductases may include thioredoxin or the like involved in a cascade reaction. Amino acid isomerase such as transglutaminase or the like is also known as an enzyme modifying protein structures.
For example, rapid separation of neuraminidase from a mixture solution of glycoprotein and neuraminidase can be performed in combinations of alcohol fractionation, salting-out, ion-exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, or affinity chromatography described above, but stable purification method of the objective protein by using only these methods is extremely limited.
It can be also considered that an enzyme inhibitor (divalent cations such as copper, sialic acid and derivatives thereof, sialyllactose, oligosialic acid, polysialic acid, Tamiflu, BCX-1812, sialidase neutralizing antibody, or the like.) is used to inhibit the enzymatic activity of neuraminidase for purification. However, it is not convenient to use a large amount of these inhibitors in the preparation process, and during purification process, there is a limitation in removing them to a concentration with no toxicity.
As such, many compositions such as a culture fluid containing the objective protein or the like include a plurality of impurities that damage the structure or stability of protein as trace elements. In order to obtain the protein with the desired quality, these impurities are required to be removed in a safe, rapid and efficient manner.
Further, the objective protein degraded or modified by the above described impurities or the objective protein biologically inactivated itself can be also impurities. In the case of glycoproteins containing sugar chains, the sugar chains are known to be greatly involved in physiological activity, stability, in vivo kinetics, solubility or the like.
When glycoproteins are produced using animal cells that are prepared by application of genetic recombination technique, neuraminidase released from dead cells are included in the obtained culture fluid. Therefore, sialic acid is removed from sugar chains of the objective glycoproteins. When sialic acid is eliminated from the glycoprotein, the galactose residue exposed to the non-reducing-end is captured by an asialoglycoprotein receptor (galactose receptor) localized in the liver and quickly degraded (Non-Patent Document 2).
As such, when the number of sialic acids bound at the terminal sugar chain is low, the blood half-life of glycoproteins is reduced, and their physiological activities cannot be sufficiently exerted. It has been also known that biological activity of Erythropoietin or the like depends on the number of sialic acids bound to the sugar chain.
Thus, because glycoproteins in which sialic acids are eliminated or glycoproteins in which the number of bound sialic acids is degradated may become impurities, it is necessary to remove neuraminidase in order to prevent elimination of sialic acid or to remove proteins having a lower number of bound sialic acids, as described above.
It is very difficult and challenging to control the composition or content of the sugar chains of proteins by chromatography. Thus, there is a demand to develop a method for purifying glycoproteins in a high purity while maintaining their sugar chains in the desired quality.
In another case, the objective protein denatured during the preparation process can be also impurities in itself. For example, aggregates (or associates, aggregates, dimers, oligomers, aggregates, multimers) resulting from denaturation of the objective proteins during the preparation process are problematic in terms of reduction of biological activity, change in in vivo kinetics, antigenicity or the like.
For example, in the case of Protein S, generation of aggregates cannot be inhibited by the typical purification method, and a purification method of generating no aggregates has not been known yet (Non-Patent Document 3). Further, a purification method for efficiently reducing the content of a cleaved form (a nicked form) has not been developed. For this reason, it is required to establish a purification method capable of removing impurities derived from the objective protein, such as aggregates or cleaved forms, in order to a sufficiently low level during purification process.
Under this background, there is a need for a method for easily purifying a protein with a desired quality by removing impurities related to the protein quality.
Meanwhile, various modes of chromatography carriers has been developed as a technology for purifying the objective protein from the protein composition in a high purity. Of them, a mixed mode carrier (or multimode carrier) is a chromatography carrier prepared by immobilizing ligands of mode of having two or more characteristics onto a single carrier, which has been recently developed.
The mixed mode carrier is known to have a unique separation property as well as that of combined two modes, and has been a useful tool for protein purification (Non-Patent Document 4).
However, there has been no report on a standard method for purifying other proteins, except for antibodies, using the mixed mode carrier. The mixed mode carrier such as Capto adhere (manufactured by GE Healthcare) having an anion exchange group and a hydrophobic interaction group, is known only as a subsequent purification following a rough purification process of the antibody composition using Protein A chromatography, or the like (Patent Documents 1 to 3 and Non-Patent Document 5).
In this case, impurities to be removed are mainly host cell-derived proteins (HCP) or DNA, and quality of the objective protein itself cannot be changed or the ingredients thereof cannot be controlled (Non-Patent Documents 6 and 7).
Further, the mixed mode carrier such as Capto MMC (manufactured by GE Healthcare) having a cation exchange group and a hydrophobic interaction group, is known as a method for recovering the objective protein from a high-salt composition (Non-Patent Documents 8 to 9), and is also used in antibody purification (Patent Document 4). However, there are many limitations in the use in the first purification step for removing impurities related to the protein quality, controlling the ingredient, and obtaining the protein with the desired quality.
In particular, there is no report on a method for directly recovering and controlling sialic acids binding to sugar chains of proteins, aggregates or fragments of the protein itself by the first chromatography. Further, there are no reports on a method for purifying acidic proteins using a mixed mode carrier having a cation exchange group and a method for purifying basic proteins using a mixed mode carrier having an anion exchange group.